Visualization of cristae and mtDNA interactions via STED nanoscopy using a low saturation power probe

Mitochondria are crucial organelles closely associated with cellular metabolism and function. Mitochondrial DNA (mtDNA) encodes a variety of transcripts and proteins essential for cellular function. However, the interaction between the inner membrane (IM) and mtDNA remains elusive due to the limitations in spatiotemporal resolution offered by conventional microscopy and the absence of suitable in vivo probes specifically targeting the IM. Here, we have developed a novel fluorescence probe called HBmito Crimson, characterized by exceptional photostability, fluorogenicity within lipid membranes, and low saturation power. We successfully achieved over 500 frames of low-power stimulated emission depletion microscopy (STED) imaging to visualize the IM dynamics, with a spatial resolution of 40 nm. By utilizing dual-color imaging of the IM and mtDNA, it has been uncovered that mtDNA tends to habitat at mitochondrial tips or branch points, exhibiting an overall spatially uniform distribution. Notably, the dynamics of mitochondria are intricately associated with the positioning of mtDNA, and fusion consistently occurs in close proximity to mtDNA to minimize pressure during cristae remodeling. In healthy cells, >66% of the mitochondria are Class III (i.e., mitochondria >5 μm or with >12 cristae), while it dropped to <18% in ferroptosis. Mitochondrial dynamics, orchestrated by cristae remodeling, foster the even distribution of mtDNA. Conversely, in conditions of apoptosis and ferroptosis where the cristae structure is compromised, mtDNA distribution becomes irregular. These findings, achieved with unprecedented spatiotemporal resolution, reveal the intricate interplay between cristae and mtDNA and provide insights into the driving forces behind mtDNA distribution.

Fluorescence quantum yield measurements.The fluorescent quantum yields for Probe Si-rhodamines were measured in DMSO using Alexa Fluor 647 (Φ = 0.27, in PBS) as the standard substance at an excitation wavelength of 646 nm, and the quantum yields were calculated using the following equation: Φs = Φr (ArFs/AsFr)(ns 2 /nr 2 ), where s and r denote sample and reference, respectively, A is the absorbance, F is the relative integrated fluorescence intensity, and n is the refractive index of the solvent.(1 Watt on sample).Absorption spectra were measured after irradiation, and relative absorbance at the maximum wavelength was plotted as a function of irradiation time with a laser.
Phototoxicity assay.Live COS7 cells were labeled with HBmito Crimson at a concentration of 500 nM.The cells were then exposed to light at an intensity of 1.4 W/cm 2 for a specific duration.Then the cells recovered in the incubator for 30 min.Following the light exposure, the cells were treated with Calcein AM.Subsequently, the cells that lacked a green signal were identified as dead cells and quantified.The experimental protocol involved analyzing hundreds of cells using a 20× objective lens.

Cytoplasmic calcium level measurements. Cells were first incubated in HBSS solution
containing 2 μM Fluo4 for 20 minutes, followed by a subsequent 20-minute incubation in new HBSS solution.Subsequently, 500 nM HBmito Crimson was added and imaged.
Ionomycin (IO) was employed as a positive control for Ca 2+ release experiments.IO acts as a selective Ca 2+ ionophore, making the cellular membranes highly Ca 2+ permeable, inducing a rapid surge in cytoplasmic Ca 2+ concentration.IO diluted to 20 μM in imaging buffer was added to COS7 cells.The calcium response of the cells was monitored both before and after the administration of IO.
The image results were obtained on a STEDYCON microscope equipped with a 100×/1.42N.A. oil lens.The duration of a frame is about 12s, and the total time is about 12min.The Fluo4 signal was imaged in the first frame of each imaging cycle with the 488nm laser.The Confocal irradiation experiment was filmed in confocal mode with the same other parameters as the STED irradiation experiment.

Supplementary Chemical Synthesis
Materials and characterization.All chemicals used for synthesis were purchased from commercial suppliers and applied directly in the experiment without further purification.
Solvents were either employed as purchased or dried according to procedures described in the literature.The progress of the reaction was monitored by TLC on pre-coated silica plates (GF-254, 250 μm in thickness), and spots were visualized by UV light.Qingdao ocean silica gel (100-200 mesh) was used for general column chromatography purification. 1H NMR and 13 C NMR spectra were recorded on Q.One AS 400 or Bruker 600 spectrometer with CDCl3, DMSO-d6 or CD3OD as solvent.Chemical shifts are reported in parts per million relative to internal standard tetramethylsilane (Si(CH3)4 = 0.00 ppm).High-resolution mass spectra (HRMS) were obtained on a XEVO-G2QTOF (ESI) (Waters, USA) or TSQ Quantum Ultra (ESI) (Thermos, Germany).
Synthesis of compound 2. In a nitrogen-flushed flask fitted with a double port reaction bottle, compound 1 (360 mg, 1.5 mmol) was dissolved in anhydrous THF (10 mL) and the solution was cooled to -78 °C.1.3 M Lithium bis(trimethylsilyl)amide (LiHMDS) 2.5 mL, 3.3 mmol) was slowly added dropwise via a syringe to the above solution in an N2 atmosphere.After that, the reaction solution was stirred for 20 minutes at -78 °C, then warmed to room temperature, and further stirred for 5 min.After further cooling to -78 °C, dimethyldichlorosilane (TMSCl) (359 mg, 3.3 mmol) dissolved in anhydrous THF was slowly added into the system, then the solution was warmed to room temperature and stirred for 16 h.The solvent was evaporated at reduced pressure to obtain intermediate 2 without separation, which was used directly for the next step.

Fig. S3
The cytotoxicity of probe HBmito Crimson using a standard MTT assay.
Photon-stability measurements.The photon-stability of probe HBmito Crimson (10 μM)and Alexa Fluor 647 in different solutions including H2O, NaCl solution (10 mM) and artificial lipid membrane solution (2 mM) were irradiated with 1 Watt 660 nm LED laser

Fig. S4 a
Fig. S4 a Brightness comparison of HBmito Crimson and PK Mito Orange in living cell at different excitation powers (shown in the same range).b Power comparison at the

Fig. S6
Fig. S6 STED (a) and eliminate STED secondary excitation (b) images of living COS7 cell mitochondria labeled with HBmito Crimson.(c) The signal intensity profile crossed the cristae (indicated with arrow) in Fig. S6a.(d) The signal intensity profile crossed the cristae (indicated with arrow) in Fig. S6b.Scale bar 1 μm.

Fig
Fig. S8 (A) Cytoplasmic Ca 2+ -level response of HBmito Crimson-labeled cells under confocal and STED illumination.(B) Results for the positive control with ionomycin (IO) treatment.

Fig. S9
Fig. S9 The 3D-SIM results showed the distribution of mtDNA in mitochondria.Scale bar 1 μm.

Fig. S10
Fig. S10 Widefield and SIM results of HBmito Crimson-labeled IM and SYBR Goldlabeled mtDNA.a, b, c and d scales are 5 μm, and enlarged images are 1 μm.

Fig. S12
Fig. S12 Spatial changes in mtDNA and cristae at the mitochondrial tip by STED imaging.The white boxed area shows cristae remodeling.Scale bar 0.5 μm.

Fig. S15
Fig. S15 Fission process of mitochondria.The yellow arrow indicates the fission site.Scale bar 1 μm.

Fig. S18
Fig. S18 Frequency distribution histogram of mtDNA area between the control and erastintreated groups.

Table S2
Fluorescence (confocal and STED)microscopy data acquisition parameters.